Purification of a nuclear trans-acting factor involved in the regulated transcription of a human immunoglobulin heavy chain gene

The expression of the rearranged human immunoglobulin gamma 1 heavy chain gene (HIG1) was shown to be induced through its enhancer by the positive regulatory trans-acting factor(s) that was contained only in cells of B lineage. The trans-acting factors were purified from mouse myeloma NS1 cells, and HIG1-inducing activity was found mainly in fractions of molecular weight 53-127 kd and in a fraction eluted from a heparin-Sepharose column with 0.5 M KCI. This semipurified fraction contained proteins binding to the conserved octamer sequence, ATGCAAAT, in the promoter region, as well as to sequences in the enhancer region. The 0.5 M KCI eluates from a heparin-Sepharose column were applied to a DNA affinity column of synthetic oligonucleotides of the octamer sequence and the sequence TATTTTAGGAAGCAAA in the HpaII-BgIII region of the HIG1 gene enhancer. The protein eluted from the enhancer sequence-specific DNA affinity column showed a strong inducing activity for the HIG1 gene, and the molecular weight of a predominant protein was 96 kd.     

Expression of a recombinant murine IgE in transfected myeloma cells

We constructed a recombinant gene encoding an immunoglobulin (Ig) heavy chain consisting of the variable region from the phosphorylcholine (PC)-specific secreting myeloma MOPC167 and the epsilon constant region from SJL mice. This gene, cloned into the shuttle vector pSV2gpt, was transfected into J558L myeloma cells, and stable transformants that expressed the epsilon gene were cloned. The IgE heavy chain in these transformants is associated with the endogenous lambda light chain and is secreted as an intact IgE molecule. However, the secreted IgE does not bind to PC conjugated to bovine serum albumin (PC-BSA). The MOPC167 kappa chain gene was cloned into the shuttle vector pSV2neo and was transfected into the epsilon heavy-chain transformant. Stable transformants were cloned that expressed both the epsilon heavy chain and the kappa light chain. IgE secreted from such a transformant was shown to bind to PC-BSA. Both types of secreted recombinant IgE bound to rat basophilic leukemia (RBL) cells, but only the IgE produced by the cell line transformed with the MOPC167 kappa gene could be cross-linked with PC-BSA to cause serotonin release.     

Enhancer sequences located 3' of the mouse immunoglobulin lambda locus specify high-level expression of an immunoglobulin lambda gene in B cells of transgenic mice

In contrast to the mouse immunoglobulin heavy chain and kappa light chain genes, very little is known about the regulation of expression of the immunoglobulin lambda light chain locus. To identify elements responsible for lambda gene regulation we mapped DNaseI hypersensitive sites associated with a functionally rearranged lambda 1 gene in nuclei from the myeloma cell line J558L. Tissue-specific hypersensitive sites were identified 2.3 to 2.5 kb upstream of the CAP site of both the lambda 1 gene and the unrearranged variable (V) lambda 2 gene segments. DNA sequences flanking the lambda 1 gene were isolated and tested for their influence on expression of the lambda 1 gene after transfection into myeloma cells and after injection into fertilized mouse eggs. Two enhancer elements were identified downstream of the lambda 1 gene. A proximal element (located 4 to 10 kb 3' of the gene) enhanced expression of a lambda 1 gene in stable myeloma cell transfectants but had no effect on the expression of a heterologous reporter gene in transient assays. A second, distal element, located approximately 30 kb 3' of the gene, enhanced heterologous expression in J558L cells expressing a lambda gene but not in a non-lambda myeloma cell line (SP2/0-Ag14). Co-injection of cosmids containing the lambda 1 gene and both the proximal and distal downstream elements into fertilized mouse eggs resulted in high-level expression of the lambda 1 transgene in B cells of transgenic mice. The identification of these lambda regulatory elements, in addition to contributing to an understanding of lambda gene regulation per se, will facilitate the study of the regulation of differential expression of kappa and lambda light chain genes in the immune system.     

Genes activated in the presence of an immunoglobulin enhancer or promoter are negatively regulated by a T-lymphoma cell line.

The tissue-specific expression of immunoglobulin genes can be partially explained by a requirement for activating factors found only in B lymphocytes and their derivatives. However, loss of immunoglobulin expression upon fusion of an immunoglobulin-producing myeloma cell with a T lymphoma cell (BW5147) or fibroblast (L cell) suggests that negatively acting factors also play a role in the tissue specificity of immunoglobulin genes. Expression of a cloned immunoglobulin heavy-chain gene introduced into myeloma cells was suppressed after fusion of the myeloma transformants with BW5147. The presence of either the immunoglobulin heavy-chain enhancer or promoter conferred suppression, under similar conditions, upon a heterologous gene that is normally expressed in both B and T lymphocytes. These immunoglobulin heavy-chain gene control regions, or gene modifications induced by them, are subject to negative control by T-lymphocyte-derived factors.     

Il-3-dependent mouse clones that express B-220 surface antigen, contain Ig genes in germ-line configuration, and generate B lymphocytes in vivo

The continuously proliferating clones L/B AgA2, CB/Bm 7, Ba/C1, and Bc/Bm 11 were established from bone marrow of MRL/LPR, CBA/J, and BALB/c mice. These clones carry the B cell lineage surface antigen B-220 but not antigens normally expressed on mature B lymphocytes, myeloid cells, or T lymphocytes. Their immunoglobulin mu heavy chain and kappa light chain genes are in germ-line configuration. The G418 resistance gene was introduced into each clone with a retrovirus vector and then used as a selective marker for the progeny of transfected cells. Clones L/B AgA2, CB/Bm 7, and Bc/Bm 11, but not Ba/C1, could develop into antibody-secreting cells after in vivo transfer. None gave rise to cells responsive to polyclonal T cell activators, nor did any differentiate into cells that could develop into granulocyte/macrophage-colony-forming cells in vitro. All grew in interleukin 3 but not in other cytokines. We conclude that clones L/B AgA2, CB/Bm 7, and Bc/Bm 11 are early precursors of B lymphocytes.     

A transgenic immunoglobulin mu gene prevents rearrangement of endogenous genes

Transgenic mice containing a microinjected rearranged immunoglobulin (Ig) mu heavy chain gene were examined for the effects on DNA rearrangement of the endogenous Ig genes. Abelson murine leukemia virus (A-MuLV) cell lines were isolated from pre-B cells of transgenic mice and of normal littermates. Microinjected mu gene RNA and a mu heavy chain protein were synthesized in every transgenic A-MuLV cell line. Only 10% of normal mouse A-MuLV transformants synthesized mu protein. A germ-line JH allele was observed in 40% of the transgenic lines, demonstrating that the block to endogenous Ig DNA rearrangement occurred at the first step of heavy chain DNA joining. All alleles were rearranged in normal mouse A-MuLV lines. Germline JH alleles were also detected in 10% of the transgenic hybridomas derived from proliferating B cells. Our results support a model of active prevention of rearrangement by the product of successfully rearranged mu genes.     

Identification of enhancer sequences 3' of the rabbit Ig kappa L chain loci

The rabbit is useful for studies of Ig L chain gene expression because of a great disparity in expression of two isotypic forms of the kappa L chain. Normally, K1 is expressed at high levels and K2 is almost silent; expression of K2 increases in mutant or experimentally allotype-suppressed animals. The reasons for the preferential utilization of the K1 isotype have not been fully elucidated. We were interested in looking for second enhancers 3' of the C kappa genes because the absence of a 3' enhancer in the K2 locus could explain the preferential utilization of the K1 isotype. However, we found a strong region of enhancer activity about 7 kb downstream of the C kappa 2 gene. Sequences in this region are highly conserved between rabbit, man, and mouse. There also appears to be a homologous 3' enhancer region in the rabbit K1 locus. We also confirmed earlier reports that the rabbit K1 intron enhancer is inactive in transient transfections into mouse B cells but find that the same construct has low but significant activity in a human B cell line. In a comparable construct the K2 intron enhancer is without activity suggesting possible differential activity of the intronic enhancers.